Myocardial fast transient K+ (Ito,f) currents contribute to early repolarization, plateau potentials, regional differences in action potential waveforms and to the generation of normal cardiac rhythms. Alterations in Ito,f densities and properties are evident in a variety of cardiovascular diseases and are associated with increased risk of arrhythmias and sudden death. The mechanisms involved in Ito,f regulation in the normal heart and Ito,f remodeling in cardiac disease, however, remain poorly understood. Considerable evidence suggests that Ito,f channels function in macromolecular protein complexes, comprising voltage-gated K+ (Kv) pore-forming () subunits of the Kv4 subfamily, together with accessory and regulatory proteins that affect channel stability, trafficking and biophysical properties. Indeed, studies in heterologous cells suggest roles for multiple accessory and regulatory proteins in the generation of Kv4-encoded channels. It has become increasingly apparent, however, that many accessory subunits interact with multiple Kv, as well as non-Kv, subunits when over- expressed in heterologous cells, clearly suggesting that studies focused on defining the physiological roles of accessory and regulatory proteins need to be completed on myocytes. The goals of the studies here are to define the physiological roles of the KChIP2, DPP6, MiRP1 and MiRP2 accessory subunits and of Ca2+/calmodulin- dependent protein kinase II (CaMKII)-mediated phosphorylation in the generation of native Ito,f channels in adult (mouse and canine) ventricular myocytes. Experiments in aim #1 will pursue recent findings suggesting critical roles for KChIP2-Kv4 protein-protein interactions in controlling the stability and the cell surface expression of Kv4-encoded Ito,f channels and delineate the mechanisms underlying KChIP2-mediated regulation of Ito,f. Experiments in aim #2 will test directly the hypotheses that accessory DPP6, MiRP1 and MiRP2 subunits function to control the expression and properties of native ventricular Kv4-encoded Ito,f channels and define the molecular mechanisms underlying DPP6-, MiRP1- and MiRP2-mediated effects. Further experiments, in aim #3, will identify CaMKII phosphorylation sites on Ito,f channel subunits and define the functional consequences of phosphorylation at these sites on the cell surface expression and the biophysical properties of native Kv4- encoded ventricular Ito,f channels. Molecular genetic strategies, in combination with biochemistry, electrophysiology and proteomics, will be exploited to accomplish these aims. It is anticipated that these studies will provide fundamentally important new insights into the molecular mechanisms that regulate the cell surface expression and the properties of native mammalian myocardial Ito,f channels. These insights will guide future efforts to define the molecular, cellular and systemic mechanisms involved in the dynamic regulation of Ito,f channels in the normal heart and the alterations in Ito,f channel expression and functioning associated with cardiac disease. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels control the heights and durations of myocardial action potentials and contribute importantly the generation of normal cardiac rhythms. Changes in Kv channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life- threatening cardiac arrhythmias. Accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising pore-forming () subunits and a variety of accessory subunits and regulatory proteins that affect channel stability, trafficking an biophysical properties. The physiological roles of Kv channel accessory and regulatory proteins, however, are poorly understood. Exploiting molecular genetic approaches in vivo and in vitro, this research proposal is focused on defining the physiological role(s) of Kv channel accessory subunits in the generation of native cardiac Kv channels and in the regulation of myocardial membrane excitability.